Ophthalmic solution of difluprednate

ABSTRACT

The present invention provides a method of treatment of inflammatory disorder of the eye, said method comprising administering into the eye of a person in need thereof, an aqueous solution comprising difluprednate as the sole active ingredient at a concentration of 0.02% to 0.04% weight by volume in an aqueous vehicle, wherein the solution is free of oil and wherein the solution is administered twice-a-day. The present invention also provides an ophthalmic solution comprising a. therapeutically effective concentration of difluprednate, a crystal growth inhibitor and pharmaceutically acceptable amounts of a solubilizer comprising a mixture of i. quaternary ammonium compound and ii. polyethoxylated castor oil, b. in an aqueous vehicle. wherein the crystal growth inhibitor is polyvinyl alcohol or its derivatives.

FIELD OF THE INVENTION

The present invention relates to an ophthalmic aqueous solution ofdifluprednate or its pharmaceutically acceptable salt and its use in thetreatment of inflammatory disorder of the eye.

The present invention relates to a method of treating inflammatorydisorder of the eye, said method comprising administering into the eyeof a patient in need thereof, an ophthalmic aqueous solution ofdifluprednate.

BACKGROUND OF THE INVENTION

Difluprednate is an anti-inflammatory corticosteroid drug represented byformula I below.

Difluprednate, a steroidal drug is practically insoluble in aqueousvehicle. The currently marketed formulation of difluprednate is anemulsion dosage form of difluprednate, approved and marketed in theUnited States under the brand name of DUREZOL®. Durezol® is anophthalmic emulsion formulation of difluprednate which comprises 0.05%w/v difluprednate emulsified between castor oil and water. It is not aclear aqueous solution. The U.S. Pat. No. 6,114,319 (herein afterreferred to as the '319 patent) is listed in the “Orange Book: ApprovedDrug Products with Therapeutic Equivalence Evaluations” against Durezol®product and describes an emulsion formulation of difluprednate whichcontains an oil and an emulsifier. Durezol® emulsion formulation isindicated for the treatment of inflammation and pain associated withocular surgery and endogenous anterior uveitis when administered fourtimes a day. As the emulsion needs to be instilled four times-a-day,there are high chances of patient non-compliance and missing a dose. Theprior art formulations of difluprednate does not provide prolongedaction. Further, it has been reported and also noted in the approvedlabel of Durezol® that the most common adverse reactions in subjectsexposed to Durezol®, (occurring in 5-10% of subjects), include blurredvision, eye irritation, eye pain, headache, increased TOP, iritis,limbal and conjunctival hyperemia, punctate keratitis. Thus, there liesa need for a formulation of difluprednate that is devoid of these sideeffects and which overcomes the existing drawbacks.

SUMMARY OF THE INVENTION

The present invention provides an aqueous solution comprising

-   -   a. therapeutically effective concentration of difluprednate, a        crystal growth inhibitor and pharmaceutically acceptable amounts        of a solubilizer comprising a mixture of        -   i. quaternary ammonium compound and        -   ii. polyethoxylated castor oil,    -   b. in an aqueous vehicle.    -   wherein the crystal growth inhibitor is polyvinyl alcohol or its        derivatives.

The present invention in another aspect, provides a method of treatinginflammatory disorder of the eye, said method comprising administeringinto the eye of a person in need thereof, an aqueous solution comprisingdifluprednate as the sole active ingredient at a concentration of about0.02% to 0.04% weight by volume, wherein the solution is free of oil andwherein the solution is administered twice-a-day.

DETAILED DESCRIPTION OF THE INVENTION

These “aqueous solution” as stated herein, is a solution ofdifluprednate in aqueous vehicle, wherein difluprednate is in thesolubilized form and not in particulate form, either microparticulate ornanoparticulate or in micellar form.

The term ‘crystal growth inhibitor’ as used herein means the additionalexcipients that prevent the difluprednate from being precipitated orcrystallized out from the aqueous vehicle. The screening for inhibitoryeffect of crystal growth of difluprednate may be carried out by physicalobservation as well as by determining the clarity of the aqueoussolution, immediately upon formulating or on storage. The solutions showpercentage transmission greater than 90%, more preferably greater than95%. When light is allowed to pass through the ophthalmic solution, thepercentage of incident light which is transmitted through the solutionis referred to as “percent transmission”. Generally, the percentagetransmission is determined at a wavelength of about 650 nm, but anyother suitable wavelength may be selected for determining the clarity ofthe solution. The aqueous solution of the present invention show percenttransmission greater than 90%, more preferably greater than 95%, morepreferably greater than 99%. The aqueous solution remains clear and freefrom particles, crystals or precipitate, upon long term storage attemperatures between 2° C. to 30° C. for a period of 6 months or more.

The aqueous solution of the present invention is free of oil. The term‘oil’ as used herein refers to oils which are hydrophobic compounds. Theexamples of the ‘oil’ include, but are not limited to triglycerides suchas, castor oil, peanut oil, arachis oil, mineral oil and the like. Theterm ‘oil’ does not include amphiphilic compounds or surfactantsobtained by derivatising oil with a hydrophilic entity such as forexample polyethoxylated castor oil.

The ophthalmic aqueous solution of the present invention comprises

-   -   a. therapeutically effective concentration of difluprednate, a        crystal growth inhibitor and pharmaceutically acceptable amounts        of a solubilizer comprising a mixture of        -   i. quaternary ammonium compound and        -   ii. polyethoxylated castor oil,    -   b. in an aqueous vehicle.    -   wherein the crystal growth inhibitor is polyvinyl alcohol or its        derivatives.

The ophthalmic aqueous solution used according to the present inventioncomprises difluprednate as the sole therapeutically active ingredient.The word ‘difluprednate’ as used herein includes prodrugs ofdifluprednate wherein the hydroxyl group in difluprednate is convertedto a labile ester or an amide. In one embodiment, the ophthalmic aqueoussolution according to the present invention does not includepovidone-iodine or any other active ingredient and is always the soleactive ingredient. The concentration (% weight by volume) ofdifluprednate is expressed in terms of difluprednate base. It is presentat a concentration that ranges from about 0.005% to 0.07% weight byvolume, preferably from about 0.02% to 0.045% weight by volume, such asfor example 0.025, 0.03, 0.035, 0.036, 0.037, 0.038, 0.039 or 0.04%,0.041%, 0.042%, 0.043% weight by volume, more preferably from about0.02% to 0.04% weight by volume.

The ophthalmic aqueous solution according to the present inventioncomprises solubilizer which is a mixture of quaternary ammonium compoundand polyethoxylated castor oil. It was found that when individualsolubilizer i.e. quaternary ammonium compound alone or polyethoxylatedcastor oil alone were used, the attempts to solubilize difluprednatewere not successful and difluprednate precipitated from the solution,either immediately or upon storage. Surprisingly, when the mixture ofthese two category of solubilizers is used, clear aqueous solution wasobtained i.e. there was no precipitation of difluprednate uponpreparation or on storage.

The quaternary ammonium compound is selected from, but not limited to,benzalkonium chloride, myristyl gamma picolinium chloride, benzethoniumchloride, benzododecinium bromide, cetalkonium chloride, cetylpyridiniumchloride, cetrimonium, tetraethylammonium bromide, polyhexamethylenebiguanide, oleyl amine and the like. In some preferred embodiments, thequaternary ammonium compound is selected from benzalkonium chloride andmyristyl gamma picolinium chloride. The quaternary ammonium compound isused in the ophthalmic solution in amounts ranging from about 0.0002% to0.1% weight by volume, preferably from about 0.002% to about 0.08%weight by volume, such as for example 0.003, 0.004, 0.005, 0.006, 0.007,0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.07% weight byvolume. According to one particular preferred embodiment, the quaternaryammonium compound is benzalkonium chloride and is present in theophthalmic aqueous solution in an amount ranging from about 0.0002% to0.08% weight by volume, preferably from about 0.0002% to 0.05% weight byvolume, more preferably from about 0.005% to 0.05% weight by volume,more preferably from about 0.01% to 0.05% weight by volume. Thepolyethoxylated castor oil that is used as a solubilizer according tothe present invention is also known by other terms like polyoxyl castoroil, or polyoxyethylene castor oil. It is marketed under varioustradenames such as Cremophor®, Acconon®, Arlatone®, Eumulgin®, Etocas®,Jeechem®, Hetoxide®, Nikkol®, Croduret®. The polyethoxylated castor oilsor polyoxyethylene castor oil derivatives that may be used in theophthalmic aqueous solutions of the present invention are described in“Handbook of Pharmaceutical Excipients”, fifth edition, 2006, page572-578. The ophthalmic aqueous solution of the present inventionpreferably comprises polyoxyl 35 castor oil, marketed under thetradename Cremophor® EL by BASF Corp.; polyoxyl 40 castor oil marketedunder the tradename Croduret®40 or Etocas®40, polyoxyl 60 castor oilmarketed under the tradename Jeechem® CA-60; polyoxyl 15 castor oilmarketed under the tradename Jeechem® CA-15 or Acconon® CA-15. Thepolyethoxylated castor oils are used in the ophthalmic aqueous solutionof the present invention in pharmaceutically acceptable amounts. Thepharmaceutically acceptable amount of ‘polyethoxylated castor oil’ranges from about 1.0% to 10.0% weight by volume. Preferably, thepolyethoxylated castor oil is present in the ophthalmic aqueous solutionin an amount ranging from about 1.5% to 6.0% w/v, such as for example2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, or 5.5% weight by volume of thesolution. According to one particular preferred embodiment, the‘polyethoxylated castor oil’ is polyoxyl 35 castor oil and is present inthe ophthalmic aqueous solution in pharmaceutically acceptable amountranging from about 3.0% to 5.0% w/v. In one particularly preferredembodiment, the aqueous solution comprises a mixture of benzalkoniumchloride and polyoxyl 35 castor oil as the solubilizer.

The crystal growth inhibitor present in the aqueous solution ispolyvinyl alcohol or its derivatives. Without the presence of thesecrystal growth inhibitors, difluprednate does not remain in solubilizedform in aqueous vehicle and precipitates out upon standing/storage. Thederivatives of the polyvinyl alcohol include, but are not limited to,polyvinyl alcohol-polyethylene glycol graft copolymer (marketed underthe trade name Kollicoat®), poly (vinyl alcohol co ethylene),polystyrene-polyvinyl alcohol graft co-polymer, polyvinylalcohol-polyvinylpyrrolidone graft co-polymer, polyvinyl alcohol-lacticacid graft co-polymer, polyvinyl alcohol-carregeenan-graft co-colymer,polyvinyl alcohol-polyether graft copolymer and the like and mixturesthereof. In one specific embodiment, the crystal growth inhibitor ispolyvinyl alcohol. It ranges from about 0.1% to 5.0% weight by volume,preferably from about 0.5% to 3.0%, such as for example 0.6, 0.7, 0.80.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4,2.5, 2.6, 2.7, 2.8 or 2.9% weight by volume of the solution.

The aqueous solution of the present invention may further include, otherconventional excipients such as viscosity increasing agent,preservatives, chelating agents, cosolvents, buffers and so on.

The viscosity increasing agent that can be used include, but are notlimited to, carboxymethylcellulose, hydroxypropyl methylcellulose,hydroxyethyl cellulose, hydroxypropyl cellulose, methylcellulose,hydroxyethyl starch, dextran, xanthan gum, sodium alginate, starch,sodium hyaluronate, carbopols, polyvinyl pyrrolidone and the like ormixtures thereof. In one preferred embodiment, viscosity enhancer iscarboxymethylcellulose. Suitably, in preferred embodiments, the aqueoussolution does not contain viscosity enhancer's such as polycarbophil andchitosan which have high molecular weight. The viscosity of the aqueoussolution according to the present invention generally ranges from about1 cps to 2000 cps, preferably about 2 cps to 1000 cps, more preferablyabout 1 cps to 300 cps, more preferably from about 2 cps to 200 cps. Inparticularly preferred embodiments, the viscosity of the ophthalmicaqueous solution is between about 2 cps to 30 cps. In one embodiment,the present invention provides an ophthalmic aqueous solution comprisinga therapeutically effective amount of difluprednate as the sole activeingredient and having a viscosity from 2 cps to 200 cps, wherein thesolution is effective in treating inflammatory disorder of the eye whenadministered twice-a-day.

The ophthalmic aqueous solution may further contain one or preservative,particularly when the dosage form is multiple dose and not single dose.The preservatives that may be used include, but are not limited to,benzyl alcohol, cetrimide, chlorhexidine, chlorobutanol, mercurialpreservatives like phenylmercuric nitrate, phenylmercuric acetate,thimerosal, phenylethyl alcohol, Polyquad®, stabilizedoxy-chlorocomplex, stabilized peroxides and perborates and the like. Itis also possible to include safer preservative systems and preservativeefficacy enhancers such as edetate disodium, N-lauroyl sarcosine or itssodium salt, boric acid, borates, biguanides like polyhexamethylenebiguanide, polyoxyalkylene diamine biguanide or its water-soluble salt;1,1′-hexamethylene-bis-{5-(4-chlorophenyl)-biguanide};1,1′-hexamethylene-bis-{5-(4-fluorophenyl)-biguanide}; (N,N″-bis(2-ethylhexyl)-3,12-diimino-2,4,11,13-tetraazatetra decanediimidamine; parabens(such as methyl-propyl, isopropyl and butyl-paraben), pyruvates,stabilized oxychloro compounds, sorbic acid/potassium sorbate, metalions, peroxides, amino acids such as arginine, tromethamine and mixturesthereof. According to another embodiment of the present invention, theophthalmic aqueous solution may be self preserving.

In one preferred embodiment, the ophthalmic aqueous solution uses apreservative selected from a biguanide, boric acid, N-lauroyl sarcosineor mixtures thereof. In one preferred embodiment, the ophthalmic aqueoussolution comprises polyhexamethylene biguanide in an amount ranging fromabout 0.001% to 0.04% w/v, preferably from about 0.002% to 0.02% w/v,such as for example 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009,0.01 or 0.02% w/v. In one preferred embodiment, the ophthalmic aqueoussolution comprises boric acid in an amount ranging from about 0.05% to1.5% w/v, preferably from about 0.1% to 1.0% w/v, such as for example,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9% w/v, more preferably from 0.4%to 0.7% w/v. In one preferred embodiment, the ophthalmic aqueoussolution comprises N-lauroyl sarcosine in an amount ranging from about0.001 to 0.5% w/v, such as for example 0.002, 0.003, 0.004, 0.005,0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 009, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 or 0.45% w/v, preferablyfrom about 0.01 to 0.1% w/v, more preferably from 0.02 to 0.05% w/v. Inone particularly preferred embodiment, the ophthalmic aqueous solutioncomprises a mixture of polyhexamethylene biguanide, boric acid andN-lauroyl sarcosine as preservative. In one specific embodiment, it wasfound that when benzalkonium chloride was used alone, the solution wasnot preserved effectively, which was indicated by the results of thepreservative efficacy test. When a biguanide was added to the solution,the solution was found to be preserved effectively i.e. the solutionpassed the preservative efficacy test as specified in EuropeanPharmacopoeia.

The chelating agents that can be used include, but are not limited to,edetate disodium, ethylenediamine tetracetic acid, edetic acid, disodiumedetate dihydrate, diethylenetriamine pentaacetic acid etc. A preferredchelating agent is ethylenediamine tertaacetic acid or disodium edetate.In one preferred embodiment, the ophthalmic aqueous solution comprisesdisodium edetate in an amount ranging from about 0.001% to 0.5% w/v,such as for example 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008,0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15,0.2, 0.25, 0.3, 0.35, 0.4 or 0.45% w/v, preferably from about 0.01 to0.1% w/v, more preferably in an amount of 0.03-0.07% w/v.

The pH adjusting agents and/or buffer that may be used are selectedfrom, but not limited to, acetic acid, sodium acetate, tartaric acid,sodium tartrate, citric acid, sodium citrate, hydrochloric acid, sodiumhydroxide or mixtures thereof. The osmotic/tonicity adjusting agentsthat may be used include, but are not limited to, sodium chloride,potassium chloride, sodium bromide, calcium chloride, mannitol,glycerol, sorbitol, propylene glycol, dextrose, sucrose, mannose and thelike and mixtures thereof. These solutions are characterized byosmolalities of 250-375 mOsm/kg, preferably 270-350 mOsm/kg, such as forexample 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,340 or 345 mOsm/kg. Osmolality of the solutions is adjusted by additionof an osmotic/tonicity adjusting agent.

The co-solvents that can be used, include, but are not limited to,glycerol or glycerine, propylene glycol, ethylene glycol, polyethyleneglycol, glycofurol and like. Glycerol may be present in the ophthalmicaqueous solution of the present invention in an amount ranging fromabout 0.5% w/v to about 5.0% w/v, such as for example 1.0, 1.5, 2.0,2.5, 3.0, 3.5, 4.0, or 4.5% w/v, preferably from about 1.0% w/v to about3.0% w/v of the solution.

A representative aqueous solution of the present invention comprises orconsists essentially of, or consists of the following composition:

Range Ingredient/function % weight by volume Active drug- Difluprednate0.02-0.04 A quarternary ammonium compound (as 0.0002-0.08 solubilizer 1) Polyethoxylated castor oil (as solubilizer 2) 1.5-6.0Polyvinyl alcohol or its derivative as a 0.1-5.0 Crystal growthInhibitor Aqueous Vehicle q.s to 100

According to various aspects, the ophthalmic aqueous solution accordingto the present invention comprises or consists essentially of, orconsists of the following composition:

Range Ingredients % weight by volume Difluprednate 0.02-0.04 Aquarternary ammonium compound (as 0.0002-0.08  solubilizer 1)Polyethoxylated castor oil (Polyoxyl 35 1.5-6.0 castor oil) Polyvinylalcohol or its derivative as a 0.1-5.0 Crystal growth InhibitorCosolvent 0.5-5.0 Aqueous Vehicle for eg. Water for injection q.s to 100

According to various aspects, the ophthalmic aqueous solution accordingto the present invention comprises or consists essentially of, orconsists of the following composition:

Range Ingredients % weight by volume Difluprednate 0.02-0.04 Quaternaryammonium compound 0.0002-0.08  Polyethoxylated castor oil 1.5-6.0Polyvinyl alcohol or its derivative as a Crystal 0.1-5.0 growthInhibitor Co-solvent 0.5-5.0 n-lauroyl sarcosine 0.01-0.1  Poly hexamethylene biguanide 0.001-0.04  Other Preservative 0.1-1.0 Chelatingagent 0.01-0.1  Buffer 0.001-0.05  Water for Injection q.s. to 100

According to various aspects, the ophthalmic aqueous solution accordingto the present invention comprises or consists essentially of, orconsists of the following composition:

Range Ingredients % weight by volume Difluprednate 0.03-0.04 Quaternaryammonium compound 0.01-0.05 Polyethoxylated castor oil 3.0-5.0 Crystalgrowth inhibitor, polyvinyl alcohol or 0.5-3.0 its derivative

A first ophthalmic aqueous solution, according to various aspects,comprises or consists essentially of, or consists of the followingComposition:

Range Ingredients % weight by volume Difluprednate 0.03-0.04 Quaternaryammonium compound 0.025 n-lauroyl sarcosine 0.03 Polyethoxylated castoroil 5.00 Polyvinyl alcohol or its derivative 1.40 Poly hexa methylenebiguanide 0.005 Other conventional excipients 0.05 to 2 Aqueous Vehiclefor eg. Water for injection q.s to 100

A second ophthalmic aqueous solution, according to various aspects,comprises or consists essentially of, or consists of the followingComposition:

Ingredients Range in (% w/v) Difluprednate 0.03 Benzalkonium chloride0.02 Polyoxyl 35 castor oil (Cremophor ® EL) 4.0 Polyvinyl alcohol orits derivative 1.4 Other conventional excipients 0.05 to 2 AqueousVehicle for eg. Water for injection q.s to 100

The present invention in another aspect, provides a method of treatmentof inflammatory disorder of the eye, said method comprisingadministering into the eye of a person in need thereof, an aqueoussolution comprising difluprednate as the sole active ingredient at aconcentration of 0.02% to 0.04% weight by volume in an aqueous vehicle,wherein the solution is free of oil and wherein the solution isadministered twice-a-day.

The inflammatory disorder of the eye may be one or more of the pain andinflammation associated with ocular surgery and uveitis. In particularaspect, the present invention provides method of treating acute anterioruveitis or chronic uveitis, said method comprising twice-a-dayadministration into the eye of a person in need thereof, an aqueoussolution comprising difluprednate as the sole active ingredient at aconcentration of 0.02% to 0.04% weight by volume in an aqueous vehicle,wherein the solution is free of oil. Particularly, the present inventionprovides method of treating acute anterior uveitis or chronic uveitis,said method comprising twice-a-day administration into the eye of aperson in need thereof, an aqueous solution comprising difluprednate asthe sole active ingredient at a concentration of 0.04% weight by volumein an aqueous vehicle, wherein the solution is free of oil.

Generally, the anti-inflammatory effect of the solution can bedetermined by known techniques or animal models. The inventors foundthat the method of treating acute or chronic uveitis is effective whentested in animal models as well as in human patients.

It was surprisingly found that the anti-inflammatory effect achieved bytwice-a-day administration of the aqueous solution having difluprednateat concentration of 0.03% and 0.04% weight by volume was equivalent tothe anti-inflammatory action obtained by by four-times-a-dayadministration of an emulsion formulation of the prior art, availableunder the trade name of Durezol® and having difluprednate atconcentration of 0.05% w/v. This is indeed surprisingly and unexpectedbecause the prior art composition includes significant amount of oil,moreover it contains 0.05% weight by volume of difluprednate and isinstilled four time daily as compared to the aqueous solution of presentinvention having 0.04% w/v of difluprednate, and administered twicedaily and wherein the ophthalmic aqueous solution is free of oil. Thisis all the more surprising because difluprednate being hydrophobic/oilsoluble is considered to remain in the oil phase and thus, expected toprovide better efficacy in an emulsion type of composition compared to acomposition which is aqueous based and most importantly, free of oil.The results found by the inventors are in fact contrary to theestablished hypothesis.

In one preferred embodiment, the ophthalmic aqueous solution is usefulfor the treatment of uveitis and comprises difluprednate at aconcentration ranging from about 0.02% weight by volume to 0.04% weightby volume. In one particularly preferred embodiment, the ophthalmicaqueous solution of the present invention comprises difluprednate at aconcentration of 0.03% weight by volume. In another particularlypreferred embodiment, the ophthalmic aqueous solution of the presentinvention comprises difluprednate at a concentration of 0.04% weight byvolume.

In another aspect, the present invention provides a method of treatinginflammatory disorder of the eye, said method comprising administeringinto the eye of a person in need thereof, an aqueous solution comprisingdifluprednate as the sole active ingredient at a concentration of about0.02% to 0.04% weight by volume, wherein the solution is free of oil andwherein the solution is administered twice-a-day. In other words, thepresent invention provides an ophthalmic aqueous solution for use intreatment of inflammatory disorder of the eye, the aqueous solutioncomprising difluprednate as the sole active ingredient at aconcentration of 0.02% to 0.04% weight by volume of the solution in anaqueous vehicle, the solution being free of oil, wherein the solution iseffective in treating inflammatory disorder of the eye when administeredtwice-a-day.

The present invention provides a clear aqueous solution formulation ofdifluprednate for use in treatment of inflammatory disorder of the eyewhich can be administered twice-a-day with added advantage that thesolution form enables to use lower concentration of difluprednate ascompared to the existing prior art compositions or marketed products.

The present invention provides a remarkable improvement in the method oftreatment of inflammatory disorder of the eye. By virtue of clear natureof the aqueous solution being free of any suspended particles, reducedfrequency of administration and potential for use of reduced drugconcentration, thus enhanced ocular bioavailability, the method not onlyprovides an enhanced patient compliance, but also avoids the untowardside effects such as blurred vision, irritation, foreign body sensationetc., upon instillation.

In one preferred embodiment, the ophthalmic aqueous solution ofdifluprednate is useful in the treatment of acute anterior uveitis bytwice-a-day instillation into the effected eye of the patient. Inanother preferred embodiment, the ophthalmic aqueous solution of thepresent invention is useful in the treatment of chronic uveitis bytwice-a-day instillation into the effected eye of the patient.

The inflammatory disorders of the eye that can be treated byadministering ophthalmic aqueous solution according to the presentinvention includes, but are not limited to pain and inflammationassociated with ocular surgery, uveitis, acute anterior uveitis,endogenous anterior uveitis, chronic uveitis, inflammation associatedwith ocular allergies, steroid responsive inflammatory condition of thepalpebral and bulber conjunctiva, cornea and anterior segment of theglobe such as allergic conjunctivitis, acne rosacea, superficialpunctuate keratitis, herpes zoster keratitis. In preferred embodiments,the ophthalmic aqueous solution according to the present invention isuseful in the treatment of various forms of uveitis, such as iritis oranterior uveitis, iridocyclitis and choroiditis or chorioretinitis alsoknown as posterior uveitis, acute anterior uveitis, and chronic uveitis.

In one preferred embodiment, the present invention provides a method oftreating acute or chronic anterior uveitis by twice-a-day instillationinto the effected eye of the patient, an ophthalmic aqueous solutioncomprising difluprednate at a concentration of about 0.02% to 0.04%weight by volume, wherein the solution is free of oil.

In one embodiment, the present invention provides a method of treatinginflammatory disorder of the eye, said method comprising instilling intothe eye of the person in need thereof, an aqueous solution ofdifluprednate comprising therapeutically effective concentration ofdifluprednate, a crystal growth inhibitor and pharmaceuticallyacceptable amounts of a solubilizer comprising a mixture of quaternaryammonium compound and polyethoxylated castor oil, in an aqueous vehicle,wherein the solution is free of oil.

In one embodiment, the present invention provides a method of treatinginflammatory disorder of the eye, said method comprising administeringinto the eye of a person in need thereof, an aqueous solution comprisingdifluprednate as the sole active ingredient at a concentration of about0.02% to 0.04% weight by volume, wherein the solution is free of oil andwherein the solution is administered twice-a-day, further wherein theaqueous solution comprises a crystal growth inhibitor andpharmaceutically acceptable amounts of a solubilizer comprising amixture of quaternary ammonium compound and polyethoxylated castor oil.The crystal growth inhibitor is polyvinyl pyrrolidone or its derivative,the inflammatory disorder of the eye is selected from acute anterioruveitis or chronic uveitis.

The ophthalmic aqueous solution of the present invention is capable ofenhancing the ocular bioavailability of difluprednate and thusdecreasing its frequency of administration. Whereas Durezol®, themarketed emulsion product of difluprednate is instilled four times aday, the aqueous solution of the present invention requires only twicedaily instillation to achieve the desired therapeutic effect.

In one embodiment, the present invention thus provides a method ofenhancing the ocular bioavailability of difluprednate, said methodcomprising twice-daily instillation into the eye of a person in needthereof, an ophthalmic aqueous solution of difluprednate.

In one preferred embodiment, the present invention provides a method ofenhancing the ocular bioavailability of difluprednate, said methodcomprising twice-a-day instillation into the eye of a person in needthereof, an ophthalmic solution comprising therapeutically effectiveconcentration of difluprednate, a crystal growth inhibitor andpharmaceutically acceptable amounts of a solubilizer comprising amixture of quaternary ammonium compound and polyethoxylated castor oil,in an aqueous vehicle.

The ophthalmic aqueous solution of the present invention enhance theocular bioavailability of difluprednate and reduce the frequency ofadministration to twice-a-day administration, for achieving the desiredanti-inflammatory effect, as against the repeated four times a dayadministration required for the marketed emulsion product (Durezol®).

While the present invention is disclosed generally above, additionalaspects are further discussed and illustrated with reference to theexamples below. However, the examples are presented merely to illustratethe invention and should not be considered as limitations thereto.

Example 1-4

The examples describe ophthalmic aqueous solutions of difluprednateaccording to the present invention.

TABLE 1 Ophthalmic aqueous solutions of difluprednate Quantity (% w/v)Ingredients Example 1 Example 2 Example 3 Example 4 Difluprednate 0.030.04 0.04 0.04 Benzalkonium chloride 0.02 0.02 0.02 0.025 n-lauroylsarcosine 0.02 0.02 0.02 0.03 Polyoxyl 35 castor oil 4.0  4.0 4.0 5.00(Cremophor ® EL) Polyvinyl alcohol 1.4  1.4 1.4 1.40 Carboxy methylcellulose — — 0.3 — Glycerine 2.0  2.0 2.0 1.60 Boric acid 0.50 0.5 0.50.6 Poly hexa methylene — — — 0.005 biguanide Disodium edetate 0.05 0.050.05 0.05 Sodium acetate  0.025 0.025 0.025 0.01 Acetic acid — — — 0.005Water for Injection q.s to 100 q.s to 100 q.s to 100 q.s to 100

The required quantity of benzalkonium chloride and difluprednate wastaken in a container and polyoxyl 35 castor oil (cremophor EL) wasadded. Difluprednate was solubilized in the above mixture byintermittent sonication and vortexing to form drug preconcentrate.

The required quantity of polyvinyl alcohol was dissolved in water forinjection at 70°−80° C. Other ingredients i.e. boric acid, N-laurylsarcosine, glycerine, disodium edetate, sodium acetate, acetic acid wereseparately dissolved in a portion of water for injection.

This phase was added to polyvinyl alcohol solution with stirring. Drugpreconcentrate was added to the above mixture and dissolved by mixing onmagnetic stirrer to get a solution (i). In case of example 4, anadditional step was followed wherein required quantity of polyhexamethylene biguanide was added to the solution (i). In case of example 3,an additional step was followed wherein carboxymethyl cellulosepre-mixed with a portion of water was added to the solution (i) obtainedabove, with stirring. Subsequently volume make up was carried out usingwater for injection. The solution was filtered with 0.2 micron filter.The resulting ophthalmic aqueous solutions of difluprednate (Example1-4) had a pH of about 5.0-6.0, osmolality of about 300 mOsm/kg andpercentage transmittance of about 99%. The viscosity of ophthalmicaqueous solution of Example 1, 2, and 4 was about 4-5 cps, and theviscosity of ophthalmic aqueous solution of Example 3 was about 20 cps.

The physicochemical stability of the formulations was tested uponstorage at room temperature (25° C./40% relative humidity) and at 2-8°C. for 6 months. It was found that the solutions of Example 1-4 remainedclear and free from particles, crystals or precipitate upon storage. Thepercentage transmission was greater than 95% upon storage. The assay ofdifluprednate remained in the range of 95%-105% w/v, the known andunknown impurities did not increased substantially upon storage and thecontent of impurities remained below the desired specified limit.

Comparative Examples 1-3

This example illustrates comparative non-working examples 1, 2 and 3(which are devoid of a solubilizer as per invention) and theircomparison with working Examples 5 and 6 as per Invention (whichcontains a solubilizer having a mixture of quarternary ammonium compoundand polyethoxylated castor oil). These examples illustrate thesurprising effect of use of a mixture of a quarternary ammonium compoundand a polyethoxylated castor oil (cremophor), whose combination acts asan efficient solubilizer of difluprednate in aqueous vehicle.

TABLE 2 Effect of mixture of solubilizer versus single solubilizerversus absence of solubilizer: Example of the present ComparativeExamples invention 2 3 5 6 (a) Ingredients 1 Quantity % w/v Quantity%w/v 6 (b) Difluprednate 0.04 0.04 0.04 0.04 0.04 0.04 BenzalkoniumChloride −−− 0.02 −−− 0.02 0.02 0.02 N-Lauryl Sarcosine 0.02 0.02 0.020.02 −− 0.02 Polyethoxylated castor oil −− −− 4.00 4.00 4.00 4.00(Cremophor ® EL) Polyvinyl Alcohol 1.40 1.40 1.40 1.40 1.40 −− PolyvinylAlcohol- PEG graft −− 3.00 co-polymer Glycerol 2.20 2.20 2.20 2.20 2.202.20 Water for Injection q.s to 100 Observation upon storage at roomImmediate Precipitation No precipitation till 6 temperaturePrecipitation - clear within 2 day months(−) Time Point at whichprecipitation solution not formed (+) observed (+); No precipitation (−)(+++)

Benzalkonium Chloride is a widely used component as preservative in theophthalmic formulations. However the inventors found that Benzalkoniumchloride plays an important role for the solubilisation andstabilization of difluprednate molecule in aqueous system when usedalong with a non-ionic surfactant like Cremophor®.

The inventors found that a combination of a quarternary ammoniumcompound such as benzalkonium chloride and a non-ionic surfactant suchas Polyethoxylated castor oil) (Cremophor®, solubilizes the hydrophobicdrug—difluprednate in aqueous vehicle and leads to formation of astorage stable clear aqueous solution, (working examples 5 and 6) whichsolution remains clear and there occurs no precipitation orcrystallization of difluprednate upon long term storage (at least 6months) at room temperature.

The inventors observed that use of a quarternary ammonium compound alongwith a non-ionic surfactant, Cremophor® is necessarily required forsolubilization of difluprednate. It was found that the quarternaryammonium compound along with Cremophor® plays an important role for thesolubilisation and stabilization of difluprednate molecule in aqueoussystem. Only when the two are present together, the drug getssolubilized in aqueous vehicle and there occurs no precipitation uponstorage.

However, in absence of either or both of quarternary ammonium compound(like benzalkonium chloride) or Polyethoxylated castor oil (Cremophor®),proper solubilisation of difluprednate in aqueous solution do not takeplace and the resulting formulations were unstable such thatdifluprednate gets crystallized out or precipitated out (comparativeexamples 1-3).

Comparative Examples 4-10

These comparative examples tests the effect of various crystal growthinhibitors on solubilisation and stabilization of difluprednate inaqueous solutions:

Aqueous solutions of difluprednate were prepared by substitutingpolyvinyl alcohol with other crystal growth inhibitors likehydroxypropyl methylcellulose, hydroxyl ethyl cellulose, hydroxyethylstarch, polyvinylpyrrolidone, carboxyvinyl polymer etc. and physicalstability of the solutions was studied upon storage at room temperature.The details of the quantitative formulations tested along with stabilitystudy results are presented below in Table 3:

TABLE 3 Effect of crystal growth inhibitors Comparative Examples 4 5 6 78 9 10 Category Ingredients Amount (% w/v) Anti-inflammatoryDifluprednate 0.04 Solubilizer Benzalkonium Chloride 0.02Polyethoxylated castor oil 4.00 (Cremophor ® EL) Preservative N-LaurylSarcosine 0.02 Co-solvent Glycerol 2.20 Excipients tried forHydroxypropyl methyl 0.2 − their precipitation/ cellulose crystal growthPolyvinylpyrrolidone −− 1.0 −− inhibition effect (Povidone ® K-90)Carboxyvinyl polymer −− 0.1 −− (Carbopol ®934) Hydroxyethyl starch − 1 −Guar gum −− 0.2 − Polyoxyethylene- −− 1 − polyoxypropylene co- polymer(Pluronic F-68) Vehicle Water for Injection q.s. to 100 Observation uponstorage at room temperature 5 week within 24 hours 12 days Time Point atwhich precipitation observed (+); (+) (+) (+)

The inventors of the present invention surprisingly discovered that onlya particular polymer that is, polyvinyl alcohol or its derivatives,works as efficient crystal growth inhibitor, which maintains propersolubilization of difluprednate in aqueous solution, and preventscrystallization or precipitation of drug upon long term storage at roomtemperature. (working examples 1-6 of the present invention). On theother hand, when other polymers or surfactants other than polyvinylalcohol were used, (comparative examples 4-9), it was found that thereoccurred precipitation or crystallization of drug, at varying timepoints as given in Table 3.

Example 7

Preservative Efficacy Testing: The formulation of Example 4 was testedfor efficacy of antimicrobial preservation, as per the preservativeefficacy test specified in the European Pharmacopoeia, 7.0 Edition,section 5.3.1, page 505-506.

The results of Preservative Efficacy Testing for bacteria as peracceptance criteria's A and B specified in the European Pharmacopoeiaare presented below in Table 4:

TABLE 4 Results for Preservative Efficacy Testing: PET Results -European Pharmacopoeia Criteria-A Log reduction Bacteria for complianceObservation Bacteria Log reduction At 6 Hr P. Aeruginosa NLT 2.0 5.16 S.Aureus NLT 2.0 5.05 Bacteria Log reduction At 24 Hr P. Aeruginosa NLT3.0 5.16 S. Aureus NLT 3.0 5.05 Bacteria Log reduction At 28 Days P.Aeruginosa No recovery 5.16 S. Aureus No recovery 5.05 Result as perbacteria Complies log reduction PET Results - European PharmacopoeiaCriteria-B Log reduction Bacteria for compliance Bacteria Log reductionAt 24 Hr P. Aeruginosa NLT 1.0 5.16 S. Aureus NLT 1.0 5.05 Bacteria Logreduction At 7 Day P. Aeruginosa NLT 3.0 5.16 S. Aureus NLT 3.0 5.05Bacteria Log reduction At 28 Days P. Aeruginosa No increase 5.16 S.Aureus No increase 5.05 Result as per bacteria Complies log reduction

It was observed that the aqueous solution of the present invention(example 4) comply with the specifications as per Acceptance Criteria'sA and B of efficacy of antimicrobial preservation test defined inEuropean Pharmacopoeia. i.e. required log reduction for the bacteria at6 h, 24 h and 7 day as per criteria A and 24 h, 7 day and 28 day as percriteria B was achieved. The test of efficacy of antimicrobialpreservation was also performed on another batch of aqueous solutionwhich did not contained a biguanide and it was observed that theefficacy of antimicrobial preservation was inferior to that observed inthe batch having a biguanide, inclusion of a biguanide likepolyhexamethylene biguanide helps in enhancing the anti-microbialefficacy of the aqueous solution.

Example 8

Animal efficacy study in bovine serum albumin induced chronic uveitismodel—Efficacy of difluprednate ophthalmic aqueous solution of thepresent invention was tested in bovine serum albumin induced chronicuveitis model in NZW rabbits and comparison was made with marketedDurezol® formulation. Various formulations which were tested include:

-   -   Placebo, i.e. formulation vehicle similar to Example 1 but not        having difluprednate.    -   Difluprednate ophthalmic aqueous solution of Example 1 having        0.03% w/v of difluprednate.    -   Difluprednate ophthalmic aqueous solution of Example 2 having        0.04% w/v of difluprednate.    -   Reference Item, Durezol®-Difluprednate (0.05% w/v) ophthalmic        emulsion formulation by Alcon.

The study was performed in NZW rabbits, weighing 3 to 5 kg. The animalswere divided into 7 groups, with 5 animals in each group. The 7 groupsincluded—

Group 1—Normal control group

Group 2—Water for injection (WFI) group

Group 3—BSA or bovine serum albumin (disease control) group

Group 4—Placebo group

Group 5—Example 1 group

Group 6—Example 2 group

Group 7—Reference item (Durezol®) group

On day 0, all the animals except normal control group were anesthetizedwith Ketamine and Xylazine by intramuscular route and one drop oflignocaine was applied in each eye for topical anesthesia. On day 0, ingroup 3 to 8, 200 μL of BSA (5%) was injected intravitreally in botheyes and on day 7 animals were challenged with 2.5 mL of BSA (2%)intravenous injection in marginal ear vein. On day 0, in WFI controlgroup (group 2), 200 μL of sterile WFI was injected intravitreally inthe same way mentioned above and on day 7, 2.5 mL of WFI wasadministered intravenously by marginal ear vein.

The placebo, the example 1 solution, example 2 solution and thereference item were administered after 1 hour of intravenous challengewith BSA on the day 7 to respective group of animals and theadministration was subsequently carried out till day 27 of the study. 50μL of placebo, example 1 solution and example 2 solution were instilledtopically two times at 12 hours interval to both eye of respective groupof animals using micropipette from day 7 to day 27. 50 μL of referenceitem was instilled four times at 4 hour interval to the both eye ofrespective group of animals using micropipette from day 7 to day 27. Thenormal control, WFI and BSA (disease control) animals remain untreated.On day 14, 21 and 28, each animal was anesthetized using ketamine andXylazine intramuscular injection. The eyes were examined for clinicalgrading using Zeiss slit lamp. The clinical evaluation of uveitisincluded evaluation of Total Clinical Score (on a 0 to 10 point basis asgiven in Table 5) on Day 14, 21 and 28. The clinical evaluation ofuveitis further included evaluation of Total Cell Count and TotalProtein in Aqueous Humor on Day 28.

TABLE 5 Clinical signs and grade of Uveitis Clinical Signs Grade ofUveitis (Score) Iris hyperemia Absent 0 Mild 1 Moderate 2 Severe 3Dilation of the iris and conjunctival vessels Absent 0 Mild 1 Moderate 2Cell Exudate in anterior chamber Absent 0 Mild 1 Moderate 2 Severe 3Presence of fibrinoid exudation in the pupillary area, with intenseflare in the anterior chamber Absent 0 Mild 1 Moderate 2 Total MaximumClinical Score 10

Observations: The observations of the Total Clinical Score (0-10) on day14, 21 and 28; Total Cell Count and Total Protein in Aqueous humor onday 28, are presented below in Table 6:

TABLE 6 Observations TLC (10⁴)/ml of Total Total Clinical Score (0-10)aqueous humour (pg/ml) Day 14 Day 21 Day 28 Day 28 Day 28 Protein GroupsMean SD Mean SD Mean SD Mean SEM Mean SD Normal 1.00 0.9   0.80 1.0   1.40 1.1    0.00 0.00    1.09 1.09    Control WFI 1.20 1.2   1.20 0.9   1.40 0.5    0.00 0.00    0.95 0.76    Control BSA 5.30   2.8 *** 7.601.1 *** 7.80 0.8 *** 10.75 3.65 *** 15.91 6.26 *** (Disease Control)Placebo 6.30 0.9 ns 7.70 1.6 ns   8.00 0.9 ns   12.70 4.74 ns   13.536.11 ns   Example 1 4.20 0.8 $  2.90 2.3 $$$ 3.40 2.6 $$$ 2.85 2.77 $$$3.24 2.91 $$$ group Example 2 5.10 1.3 ns 3.50 3.3 $$$ 3.30 2.6 $$$ 2.150.97 $$$ 4.09 3.57 $$$ group Reference 4.70 2.5 ns 3.60    2.2 @@@ 3.30   2.0 @@@ 3.05    1.21 @@@ 4.29    4.80 @@@ Item- Durezol ® group Totalclinical score Data were analyzed using Two way ANOVA followed byBornferroni test WFI vs BSA (Disease control); *** = p < 0.001,BSA(Disease control) vs Placebo; ns = non-significant, Placebo vsexample 1, example 2; ns = non-significant, $ = p < 0.05, $$ = p < 0.01,$$$ = p < 0.001, BSA(Disease control) vs Reference item Durezol ®; ns =non-significant, @@@ = p < 0.001 Total cell count and Total protein:Data were analyzed using One way ANOVA followed by Bornferroni test WFIvs BSA (Disease control); *** = p < 0.001, BSA (Disease control) vsPlacebo; ns = non significant, Placebo vs example 1, example 2; ns = nonsignificant; $$$ = p < 0.001, BSA (Disease control) vs Reference itemDurezol ®; @@@ = p < 0.001

It was observed that the difluprednate ophthalmic aqueous solution ofthe present invention having 0.03% w/v-0.04% w/v difluprednate (example1 and 2) when instilled twice-a-day into the eye effected by chronicuveitis, it showed a significant inhibition in total clinical score,total cell count and total protein as compared to Placebo. Particularly,in case of Example 1 group, wherein the aqueous solution has 0.03% w/vof difluprednate, the mean clinical score reduced significantly from 7.7(placebo) to 2.9 at day 21 and from 8.0 (placebo) to 3.4 at day 28.Similarly, in case of Example 2 group, wherein the formulation has 0.04%w/v of difluprednate, the mean clinical score reduced significantly from7.7 (placebo) to 3.5 at day 21 and from 8.0 (placebo) to 3.3 at day 28.

The total clinical score, total cell count and total protein levels werealso significantly attenuated by reference item Durezol® as compared toBSA (disease control) group.

The clinical score values, the total cell count and total proteincontent in aqueous humor observed at day 14, 21 and 28 by twice-a-dayadministration of low concentration (0.03% and 0.04%) difluprednateophthalmic aqueous solution of the present invention was equivalent orbetter than that observed upon four times a day administration of higherconcentration 0.05% w/v emulsion formulation Durezol (marketed referenceitem).

Example 9

Animal efficacy study in lipopolysaccharide (LPS) induced acute uveitismodel—Efficacy of Difluprednate ophthalmic aqueous solution of thepresent invention was tested in lipopolysaccharide (LPS) (an endotoxin)induced acute uveitis in female NZW rabbits and comparison was made withmarketed Durezol® formulation. Various formulations which were testedinclude:

-   -   Placebo: Formulation vehicle similar to Example 1 but not having        difluprednate.    -   Difluprednate ophthalmic aqueous solution of Example 1 having        0.03% w/v of difluprednate.    -   Difluprednate ophthalmic aqueous solution of Example 2 having        0.04% w/v of difluprednate.    -   Durezol® Difluprednate (0.05% w/v) ophthalmic emulsion        formulation by Alcon.

The study was performed in NZW rabbits, weighing 3 to 5 kg. The animalswere divided into 7 groups, with 5 animals in each group. The 7 groupsincluded—

Group 1—Normal control group

Group 2—Phosphate buffer saline (PBS) group

Group 3—Lipopolysaccharide (LPS disease control) group

Group 4—Placebo group

Group 5—example 1 group

Group 6—example 2 group

Group 7—Reference item (Durezol®) group.

On day 0, all the animals except normal control group were anesthetizedwith Ketamine and Xylazine by intramuscular route and one drop oflignocaine was applied in each eye for topical anaesthesia. On day 0, inPBS control group, 20 μL of sterile phosphate buffer saline pH-7.4 wasinjected intra-vitreally in both eyes. In group 3 to 8, 20 μL of LPS(100 ng) was injected intra-vitreally in both eyes. The placebo, theexample 1 and 2 solutions and reference item were administered 1 hourafter LPS injection, wherein 50 μL of placebo and example 1 and 2solutions were instilled topically two times at 12 hours interval toboth eye of respective group of animals using micropipette, while 50 μLof reference item was instilled four times at 4 hour interval to theboth eye of respective group of animals using micropipette. The normalcontrol, PBS and LPS (disease control) animals remain untreated. After24 hours of LPS injection, each animal was anesthetized using ketamineand Xylazine intramuscular injection. The eyes were examined forclinical grading using Zeiss slit lamp.

The clinical evaluation of uveitis included evaluation of Total ClinicalScore (on a 0 to 5 point basis as given in Table 7 below) on Day 1. Theclinical evaluation of uveitis further included evaluation of Total CellCount and Total Protein in aqueous humor on Day 1. Aqueous humor wascollected from each animal using 30 gauge needle attached withappropriate syringe after clinical scoring. Aqueous humor samples werestored at 2-8° C. till analysis. The total cell count and total proteinof each animal were calculated.

TABLE 7 Clinical signs and grade of Uveitis Clinical Signs Grade ofUveitis (Score) Iris hyperemia Absent 0 Mild 1 Moderate 2 Severe 3 PupilNormal 0 After Miosis 1 Exudate in anterior chamber Absent 0 Present 1Total Maximum Clinical Score 5

Observations: The observations of the Total Clinical Score (0-5); TotalCell Count and Total Protein in aqueous humor on day 1, are presentedbelow in Table 8:

TABLE 8 Observations Total Clinical TLC (10⁴)/ml of Total Protein Score(0-5) aqueous humour (pg/ml) Groups Mean SD Mean SEM Mean SD NormalControl 0.0 0.0    0.15 0.1  0.91 0.6  PBS Control 0.3 0.5    0.15 0.1 1.04 0.3  LPS (Disease Control) 3.1 0.7 ### 231.75 87.4 # 21.94  4.7 ###Placebo 3.0 1.1 ns   224.8   71.4 Ns 20.23   3.9 Ns Example 1 1.0 0.9$$$ 30.2 12.9 $ 9.65 8.8 $ Example 2 0.7 0.8 $$$ 29.56 18.6 $ 10.11 9.1$ Durezol ® 0.7 0.5 *** 21.2  9.8 * 13.21 8.6 * Total clinical score:PBS vs LPS (Disease control) = t-test (# = p < 0.05,### = p < 0.001),LPS (Disease control) vs Placebo = t-test; (ns = non significant);Placebo vs example 1 and 2 = One way ANOVA followed by Dunnett'sMultiple Comparison Test($ = p < 0.05, $$ = p < 0.01, $$$ = p < 0.001),;LPS (Disease control) vs Reference Durezol= t-test (* = p < 0.05, ** = p< 0.01, *** = p < 0.001); Durezol vs example 1, example 2 = One wayANOVA followed by Dunnett's Multiple Comparison Test ($ = p < 0.05, ns =non-significant)

It was observed that the difluprednate ophthalmic aqueous solution ofthe present invention having 0.03% w/v-0.04% w/v difluprednate (example1 and 2) when instilled twice-a-day into the eye effected by acuteuveitis, it showed a significant inhibition in total clinical score,total cell count and total protein as compared to Placebo. Particularly,in case of example 1 group, (0.03% w/v difluprednate solution), the meanclinical score reduced significantly from 3.0 (placebo) to 1.0 at day 1.Similarly, in case of example 2 group, (0.04% w/v difluprednatesolution), the mean clinical score reduced significantly from 3.0(placebo) to 0.7.

The total clinical score, total cell count and total protein levels werealso significantly attenuated by reference item Durezol® as compared toBSA (disease control) group. Particularly, the mean clinical scorereduced from 3.0 (placebo) to 0.7 at day 1.

The clinical score values, the total cell count and total proteincontent in aqueous humor observed at day 1 by twice-a-day administrationof low concentration (0.03% w/v and 0.04% w/v) difluprednate ophthalmicaqueous solution of the present invention was equivalent or better thanthat observed upon four times a day administration of higher strength0.05% w/v emulsion formulation Durezol® (marketed reference item).

The invention claimed is:
 1. A method of treating an inflammatorydisorder of the eye, said method comprising administering twice-a-day, aclear aqueous solution comprising, in an aqueous vehicle: a. about 0.02%w/v to about 0.04% w/v difluprednate, wherein the difluprednate is notin microparticulate form; b. about 0.5% w/v to about 3.0% w/v ofpolyvinyl alcohol or derivative thereof; and c. a solubilizer comprising(i) about 0.01% w/v to about 0.05% w/v quaternary ammonium compound and(ii) about 1.5% w/v to about 6.0% w/v polyethoxylated castor oil; intothe eye of a person in need thereof.
 2. The method as claimed in claim1, wherein the inflammatory disorder of the eye is selected from thegroup consisting of pain and inflammation associated with ocular surgeryand uveitis.
 3. The method as claimed in claim 1, wherein theinflammatory disorder of the eye is acute anterior uveitis or chronicuveitis.
 4. The method as claimed in claim 1, wherein the difluprednateis present at a concentration of 0.04% weight by volume.
 5. The methodas claimed in claim 1, wherein the difluprednate is present at aconcentration of 0.03% weight by volume.
 6. The method of claim 1,wherein the difluprednate is about 0.03% w/v to about 0.04% w/v.
 7. Themethod of claim 1, wherein the polyvinyl alcohol or derivative thereofis about 1.4% w/v.
 8. The method of claim 1, wherein the polyvinylalcohol or derivative thereof is about 3.0% w/v.
 9. The method of claim1, wherein the quaternary ammonium compound is benzalkonium chloride.10. The method of claim 1, wherein the quaternary ammonium compound isabout is about 0.025% w/v.
 11. The method of claim 1, wherein thepolyethoxylated castor oil is polyoxyl 35 castor oil.
 12. The method ofclaim 1, wherein the polyethoxylated castor oil is about 3.0% w/v toabout 5.0% w/v.
 13. The method of claim 1, wherein the polyethoxylatedcastor oil is about 5.0% w/v.
 14. The method of claim 1, wherein theclear aqueous solution further comprises a preservative selected frombiguanide, boric acid, n-lauryl sarcosine, or a combination thereof. 15.The method of claim 1, wherein solution percent transmission of theclear aqueous solution is greater than 90%.
 16. A method of treating aninflammatory disorder of the eye, said method comprising administering aclear aqueous solution comprising, in an aqueous vehicle: a. about 0.03%w/v to about 0.04% w/v difluprednate, b. about 0.5% w/v to about 3.0%w/v polyvinyl alcohol or derivative thereof, c. about 0.01% w/v to about0.05% w/v benzalkonium chloride; and d. about 1.5% w/v to about 6.0% w/vpolyoxyl 35 castor oil, into the eye of a person in need thereof. 17.The method of claim 16, wherein the method comprises administering theclear aqueous solution into the eye of a person in need thereof twice aday.